Determination of serum creatinine

ABSTRACT

A colorimetric process for the quantitative determination of creatinine in blood serum or urine wherein an alkaline pH in the approximate range of 11.0 to 13.5 is maintained during colorimetry evaluation and in which urea and a detergent are caused to react simultaneously and synergistically with the serum or urine protein to prevent extraneous chromogen formation in the conventional alkaline-picrate reaction for creatinine.

United States Patent 11 1 Jarvis [451 July 15, 1975 [73] Assignee: Coulter Diagnostics, Inc., Hialeah,

Fla.

22 Filed: Mar. 4, 1974 211 Appl. No.: 447,864

Alvar Jarvis, Miami, Fla.

[52] 'U.S. Cl. 23/230 B; 252/408 [51] Int. Cl. G0ln 33/16 [58] Field of Search 23/230 B; 252/408 [56] References Cited UNITED STATES PATENTS 3,557,018 l/1971 Scheuerbrandt 23/230 B 3.682.586 8/1972 Ertingshausen 23/230 B 3,705,013 12/1972 Dewhurst 23/230 B Primary Examiner-R. E. Serwin Attorney, Agent, or Firm-Silverman & Cass, Ltd.

[57] ABSTRACT A colorimetric process for the quantitative determination of creatinine in blood serum or urine wherein an alkaline pH in the approximate range of 11.0 to 13.5 is maintained during colorimetry evaluation and in which urea and a detergent are caused to react simultaneously and synergistically with the serum or urine protein to prevent extraneous chromogen formation in the conventional alkaline-picrate reaction for creatinine.

21 Claims, No Drawings 1 DETERMINATION OF SERUM CREATININE BACKGROUND OF THE INVENTION This invention relates to the analysis of body fluids for the quantitative determination of the creatinine content of the fluid, such as blood serum and urine.

The quantitative determination of creatinine is a very valuable procedure in connection with biologic fluids such as blood serum and urine, for the diagnosis of certain diseases. Creatinine is a waste product removed from the blood stream by the kidneys. Since creatinine is excreted only by way of the kidneys, for instance, kidney disease can be diagnosed from an increased creatinine content measured in the serum. Quantitative creatinine determination also is useful in the diagnosis of urinary obstruction, intestinal obstruction, and nephritis, an inflamation of the kidneys caused by infection, the degenerative process or vascular disease. In U.S. Pat. No. 3,705,013, there is discussed some features of critical creatinine determinations desired for accurate and effective diagnosis of diseases.

US. Pat. Nos. 3,557,018 and 3,705,013 discuss many known methods for determination of creatinine in biologic fluids.

DESCRIPTION OF THE PRIOR ART Most current methods for the determination of creatinine in serum or urine rely on the development of a red-orange color formed by the interaction of alkalinepicrate and creatinine, first reported by M. Jaffe in 1886 and applied by Otto Folin to determination of urinary creatinine in 1904. Since that time, this method using the so-called Jaffe reaction has become the most commonly used method for creatinine analysis.

The Jaffe reaction is not considered specific for creutinine in the presence of protein, glucose, and a number of unknown substances present in normal human sera. Methods designed to improve specificity have, for the most part, relied on use of protein-free filtrates. In addition to utilization of protein-free filtrates, so-called "specific methods" for creatinine have been introduced in which the filtrate is used in adsorption-elution techniques; see Brod, J. and Kotatko, J., J. Cas. Lek. Ces. 88,665 Excerpta Medica, Amst. 3,477 (1950); Hare. R. 5., Proc. Soc. Exp. Biol. N.Y., 74,148 I950); Haugen. H. N. and Blegen, E. M., Scand. J. Clin. Invest. 5.58 1953 and Polar, E. and Metkoff, J., Clin. Chem. 1 1.763 (1965). The most common adsorption media used have been hydrated aluminum silicate and ionexchange resin.

The problems encountered with adsorption-elution techniques center around complex procedures to effect the adsorption and elution, and the fact that the nonspecificity is still not completely eliminated.

Alternate specific techniques have utilized reagents such as ceric sulfate with protein-free filtrates to obtain destruction of interfering substances. See Kostir, J. V. and Rabek, V., Biochim. Biophys. Acta 5.210 1950); Kostir, J. V. and Sonka, J., Biochim. Biophys. Acta 8.86 1952) and use ofcreatinine destroying bacteria Dubos, R. and Miller, V. F., J. Biol. Chem. 121.427 (1937). The latter procedure is one in which serum protein-free filtrates are divided into two aliquots; one aliquot being treated with the creatinine destroying bacteria, the other aliquot left untreated. Subsequent Jaffe reactions with both aliquots give different adsorbance readings. Subtracting the bacteriatreated" from the non-treated aliquot value is assumed to yield a true creatinine value. Specificity cannot be satisfactorily proven by this method either.

More recently, attempts have been made to develop Jaffe reaction methods avoiding serum deproteinization which fall into two primary categories. The first involves a so-called kinetic procedure requiring critically-timed measurement of rate of formation of the reddish-orange Jaffe color represented by Larsen, K., Clin. Chim. Acta 41,209 (1972). Problems arise with the need for relatively sophisticated kinetic measurement spectrophotometers and use of extremely short color development times (30-l20 seconds). Effects of interference by non-creatinine, Jaffe-reactive chromogens cannot be entirely disproven, as appears from Raabo, E. and Walloe-Hansen, P., Scand. J. Clin. Lab. Invest. 29,297 (1972) and Heinegard, D. and Tiderstrom, 0., Clin, Chim. Acta 43,305 (1973).

The second avenue of approach consists of introduction of suppression of non-creatinine, Jaffe-positive chromogens by varying reaction pH or incorporating reagents shown to suppress the effect of color interference by non-creatinine substances. Reagent blanks or running serums at two different pHs are an integral part of all of these approaches as seen from Raabo, E. and Walloe-Hansen, P. Scand. J. Clin. Lab. Invest. 29,297 (1972); Heinegard, D. and Tiderstrom, 6., Clin. Chim. Acta 43,305 (1973). The methods relying solely on the assumption that running parallel reactions on the same serum, one at a higher pH than the other, subtracting the low pH value from that of the higher pH, and then using the factor 2.3 as a correction for protein interference, as taught by Raabo, E. and Walloe-Hansen, P., Scand. J. Clin. Lab. Invest. 29,297 (1972), based on a large number of comparisons with a previous specific method as taught by Brod, J. and Sirota, J. H., J. Clin. Invest. 27,645 (1948) leaves doubts about accuracy primarily because of this statistically observed 2.3 factor and complete reliance on specificity based on the two separate pH dependent reactions.

SUMMARY OF THE INVENTION With the above delineated problems of the prior art in mind, the invention provides a procedure for the detection of creatinine which employs an alkaline-picrate reagent which substitutes sodium phosphate for the more commonly used sodium hydroxide or lithium hydroxide. Urea is incorporated into the alkaline-picrate reagent. An aralkane sulfate detergent of sodium, potassium or lithium, such as sodium dodecyl sulfate (sodium lauryl sulfate) and sodium borate are added to the reagent. In this procedure, suppression of interfering chromogens is maximized and the serum blank is eliminated.

It is believed that in the presence of urea, the protein molecules change in configuration and unfold as a result of mild denaturation. This configuration change increases exposure of the protein molecule to binding with the sodium dodecyl sulfate with resulting decrease in blank activity. Test results using the invention have correlated well with reference methods using filtrates and adsorption techniques.

DESCRIPTION OF SPECIFIC EMBODIMENTS The following description is given to enable persons skilled in the art to more clearly understand and practice the invention. They should not be considered as limitations upon the scope of the invention but should be regarded as illustrative only.

The reagent formulations are as follows:

A. Saturated picric acid solution approximately 15 grams added to warm distilled water until no further dissolving is observed.

i. Sodium dodecyl sulfate solution 4 grams/100 ii. Sodium borate in sodium or potassium phosphate-dibasic solution 0.05 M of borate to 0.05 M phosphate.

iii. Urea solution 40 grams/100 ml.

In using these stock or prepared solutions, a first working solution is prepared consisting of two parts of the 40% urea solution combined with two parts of the 4% sodium dodecyl sulfate solution. A preferred alternate working solution consists of one part of the urea solution with three parts of the sodium dodecyl sulfate solution. Then, one part of a said sodium dodecyl sulfate urea solution is mixed with one part of the boratephosphate buffer solution and buffered using sodium hydroxide to a pH of about 12.4. The second working solution comprises the saturated picric acid solution. Prior to use, four parts of the first working solution are mixed with one part of the saturated picric acid solution to provide a single alkaline picrate reagent.

In the creatinine determination procedure using the invention, 0.01 to 2.0 ml of sample has been used with 0.2 to 25.0 ml of final mixed reagent. Preferably, however, 0.1 to 0.2 ml of sample with 4.0 ml of final reagent is used. ln this preferred procedure, 4.0 ml of alkaline picrate reagent is introduced into a suitable test tube and pre-incubated at 37 Centrigrade for 3 to 5 minutes. Then, 0.1 or 0.2 ml of sample is mixed into the reagent and incubation at 37 Centrigrade maintained for from to 30 minutes. Preferably, such incubation is maintained for minutes.

The incubated solution then is introduced into a colorimeter or spectrophotometer against a blank and light absorption of the thusly formed creatinine picrate is read at 490 to 535 nm. Preferably, the reading is made at 500 nm. Then, one can calculate the value of unknown against primary aqueous standard or secondary protein-based standard run in conjunction with the unknown using the following formula:

(1) Value of Unknown O.D. Unknown/O.D. Standard X value of standard.

The specific example given above is capable of being varied in certain respects within the parameters of the invention. The alkaline picrate reagent can be buffered to within a pH range of l 1.0 to 13.5 by selective mixing of the buffer solution, to wit, the sodium borate in the sulfate solution as mentioned. Optical density measurements can be taken within the range of wavelengths statedv In addition, other modifications may occur to the skilled artisan relating to a particular material for mulation to utilize the essential teachings of the invention. For instance, other detergents which may be used are: sodium octyl sulfate. sodium tetradecyl sulfate, sodium heptadecyl sulfate. sodium cetyl sulfate. sodium myristyl sulfate. sodium octadecyl sulfate, sodium oleyl sulfate, sodium tridecyl sulfate, potassium lauryl sulfate and dodecyl benzene sulfate. The analagous potassium or lithium salts are equally useful. Generally, the detergent may be characterized as an aralkane sulfate of sodium. potassium or lithium.

What is claimed is:

l. A colorimetric process for the determination of creatinine in a biologic fluid sample containing proteins comprising,

A. adding a reagent to the biologic fluid which forms a colored complex having a characteristic wavelength of maximum light absorption with the creatinine in said fluid, while preventing the interference of protein with the desired determination by forming protein complexes which prevent formation of extraneous chromogens of a non-creatinine character;

B. measuring the degree of light absorption at said characteristic wavelength; and

C. determining the creatinine concentration in said fluid by comparing the measured degree of light absorption with values of light absorption obtained from standard aqueous solutions containing the reagent and known creatinine concentrations.

2. The process of claim 1 wherein urea is added to the sample prior to colorimetric determinations being made in an amount sufficient to form such protein complexes.

3. The process of claim 2 wherein a detergent is added to the urea and the combination is then added to sample.

4. The process of claim 3 wherein the detergent selected is sodium octyl sulfate.

5. The process of claim 3 wherein the detergent selected is sodium tetradecyl sulfate.

6. The process of claim 3 wherein the detergent selected is sodium heptadecyl sulfate.

7. The process of claim 3 wherein the detergent selected is sodium cetyl sulfate.

8. The process of claim 3 wherein the detergent selected is sodium dodecyl sulfate.

9. The process of claim 3 wherein the detergent selected is sodium myristyl sulfate.

10. The process of claim 3 wherein the detergent selected is sodium octadecyl sulfate.

11. The process of claim 3 wherein the detergent selected is sodium oleyl sulfate.

12. The process of claim 3 wherein the detergent selected is sodium tridecyl sulfate.

13. The process of claim 3 wherein the detergent selected is dodecyl benzene sulfate.

14. The process of claim 3 wherein the detergent comprises a sodium, potassium or lithium aralkane sulfate solution.

15. The process of claim 3 wherein sample size of sample is maintained from 0.01 to 2.0 ml.

16. The process of claim 3 wherein the volume of reagent is maintained from 0.2 to 25.0 ml.

17. The process of claim 3 wherein pH is maintained from 1 1.0 to 13.5 during colorimetry.

18. The process of claim 3 wherein a buffer is added to the reagent to maintain a pH of between 1 1.0 to 13.5 during colorimetry.

19. A reagent system for use in the determination of creatinine in a biological fluid sample containing proteins and in which the creatinine concentration in the sample is determined by comparing the measured degree of light adsorption with values of light adsorption obtained from standard solutions;

6 tion of Part A is mixed with the solution of Part B to provide an alkaline-picrate reagent used in the creatinine determination.

21. The reagent system as claimed in claim 20 in which the alkaline-picrate reagent is preincubated at 37 C. and then the sample is introduced into the reagent and incubated at 37 C. for from 10 to 30 minutes. l =l 

1. A COLORIMETRIC PROCESS FOR THE DETERMINATION OF CREATIMINE IN A BIOLOGIC FLUID SAMPLE CONTAINING PROTEINS COMPRISING, A. ADDING A REAGENT TO THE BIOLOGIC FLUID WHICH FORMS A COLORED COMPLEX HAVING A CHARACTERISTIC WAVELENGTH OF MAXIMUM LIGHT ABSORPTION WIH THE CREATININE IN SAID FLUID, WHILE PREVENTING THE INTERFERENCE OF PROTEIN WITH THE DESIRED DETERMINATION BY FORMING PROTEIN COMPLEXES WHICH PREVENT FORMATION OF EXTRANEOUS CHROMOGENS OF A NON-CREATININE CHARACTER, B. MEASURING THE DEGREE OF LIGHT ABSORPTION AT SAID CHARACTERISTIC WAVELENGTH, AND C. DETERMINING THE CREATININE CONCENTRATION IN SAID FLUID BY COMPARING THE MEASURED DEGREE OF LIGHT ABSORPTION WITH VALUES OF LIGHT ABSORPTION OBTAINED FROM STANDARD AQUEOUS SOLUTIONS CONTAINING THE REAGENT AND KNOWN CREATININE CONCENTRATIONS.
 2. The process of claim 1 wherein urea is added to the sample prior to colorimetric determinations being made in an amount sufficient to form such protein complexes.
 3. The process of claim 2 wherein a detergent is added to the urea and the combination is then added to sample.
 4. The process of claim 3 wherein the detergent selected is sodium octyl sulfate.
 5. The process of claim 3 wherein the detergent selected is sodium tetradecyl sulfate.
 6. The process of claim 3 wherein the detergent selected is sodium heptadecyl sulfate.
 7. The process of claim 3 wherein the detergent selected is sodium cetyl sulfate.
 8. The process of claim 3 wherein the detergent selected is sodium dodecyl sulfate.
 9. The process of claim 3 wherein the detergent selected is sodium myristyl sulfate.
 10. The process of claim 3 wherein the detergent selected is sodium octadecyl sulfate.
 11. The process of claim 3 wherein the detergent selected is sodium oleyl sulfate.
 12. The process of claim 3 wherein the detergent selected is sodium tridecyl sulfate.
 13. The process of claim 3 wherein the detergent selected is dodecyl benzene sulfate.
 14. The process of claim 3 wherein the detergent comprises a sodium, potassium or lithium aralkane sulfate solution.
 15. The process of claim 3 wherein sample size of sample is maintained from 0.01 to 2.0 ml.
 16. The process of claim 3 wherein the volume of reagent is maintained from 0.2 to 25.0 ml.
 17. The process of claim 3 wherein pH is maintained from 11.0 to 13.5 during colorimetry.
 18. The process of claim 3 wherein a buffer is added to the reagent to maintain a pH of between 11.0 to 13.5 during colorimetry.
 19. A reagent system for use in the determination of creatinine in a biological fluid sample containing proteins and in which the creatinine concentration in the sample is determined by comparing the measured degree of light adsorption with values of light adsorption obtained from standard solutions; A. Saturated picric acid solution in distilled water; and B. i. urea solution; ii. a buffered solution of sodium borate in a phosphate salt of sodium or potassium buffered to a pH of between 11.0 to 13.5; and iii. an aralkane sulphate detergent solution.
 20. The reagent system of claim 19 in which the solution of Part A is mixed with the solution of Part B to provide an alkaline-picrate reagent used in the creatinine determination.
 21. The reagent system as claimed in claim 20 in which the alkaline-picrate reagent is preincubated at 37* C. and then the sample is introduced into the reagent and incubated at 37* C. for from 10 to 30 minutes. 